Literature DB >> 35463075

Chemical Characterization and Metabolic Profiling of the Compounds in the Chinese Herbal Formula Li Chang Decoction by UPLC-QTOF/MS.

Baofu Lin1, Shaoju Guo1, Xinxin Hong1, Xiaoyan Jiang1, Haiwen Li1, Jingwei Li1, Linglong Guo1, Mianli Li1, Jianping Chen1, Bin Huang1, Yifei Xu1.   

Abstract

Background: Li Chang decoction (LCD), a Chinese medicine formula, is commonly used to treat ulcerative colitis (UC) in clinics. Purpose: This study aimed to identify the major components in LCD and its prototype and metabolic components in rat biological samples.
Methods: The chemical constituents in LCD were identified by establishing a reliable ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF/MS) method. Afterwards, the rats were orally administered with LCD, and the biological samples (plasma, urine, and feces) were collected for further analyzing the effective compounds in the treatment of UC. Result: A total of 104 compounds were discriminated in LCD, including 26 flavonoids, 20 organic acids, 20 saponins, 8 amino acids, 5 oligosaccharides, 5 tannins, 3 lignans, 2 alkaloids, and 15 others (nucleosides, glycosides, esters, etc.). About 50 prototype and 94 metabolic components of LCD were identified in biological samples. In total, 29 prototype components and 22 metabolic types were detected in plasma. About 27 prototypes and 96 metabolites were discriminated in urine, and 34 prototypes and 18 metabolites were identified in feces.
Conclusion: The flavonoids, organic acids, and saponins were the major compounds of LCD, and this study promotes the further pharmacokinetic and pharmacological evaluation of LCD.
Copyright © 2022 Baofu Lin et al.

Entities:  

Year:  2022        PMID： 35463075      PMCID： PMC9020952          DOI： 10.1155/2022/1322751

Source DB:  PubMed          Journal:  Evid Based Complement Alternat Med        ISSN： 1741-427X            Impact factor:   2.650


1. Introduction

Traditional Chinese medicine (TCM) attracts more attention in the world since it possesses reliable therapeutic efficacy in some complex diseases, especially chronic illness [1]. The chemical composition of Chinese herbal compound is complex, and the composition of the multi-Chinese medicine is crossed, summarized as “multitarget and multicomponent,” which is the feature of TCM [2, 3]. This characteristic promotes the curative effect and reduces toxicity; however, it brings enormous challenge to figure out the effective components and mechanism for the therapeutic effect [4]. Li Chang decoction (LCD), a Chinese compound prepared from twelve Chinese medicine including Codonopsis Radix (CR), Notoginseng Radix et Rhizoma (NRR), Bletillae Rhizoma (BR), Sophorae Flos (SF), Glycyrrhizae Radix et Rhizoma (GRR), Cynanchi Paniculati Radix et Rhizoma (CPRR), Typhae Pollen (TP), Chebulae Fructus (CF), Atractylodis Macrocephalae Rhizoma (AMR), Ailanthi Cortex (AC), Coicis Semen (CS), and Halloysitum Rubrum (HR), has been commonly used to treat ulcerative colitis (UC) in clinics for over 20 years(Figure 1). UC is a chronic disease of inflammatory bowel diseases, which seriously impact the life quality of patients, and is sometimes life-threatening. LCD remarkably reduces the symptoms and recurrence rate of UC in clinical [5]. Although some of the major ingredients such as the polysaccharides from CR and AMR and rutin from SF have been proved effective in the treatment of UC, the effective components of LCD are still controversial and unclear [6-8]. Therefore, the systematic research on the effective component and metabolite profiling of LCD is an urgent need.
Figure 1

Decoction samples of 12 Chinese herbal medicines in LCD. (a) Codonopsis Radix; (b) Atractylodis Macrocephalae Rhizoma; (c) Coicis Semen; (d) Ailanthi Cortex; (e) Cynanchi Paniculati Radix et Rhizoma; (f) Halloysitum Rubrum; (g) Sophorae Flos; (h) Notoginseng Radix et Rhizoma; (i) Bletillae Rhizoma; (j) Chebulae Fructus; (k) Typhae Pollen; (l) Glycyrrhizae Radix et Rhizoma.

Ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF/MS) provides a rapid and reliable method to identify the component of natural medicine, which promotes the development of natural medicine component analysis and new drug discovery [9, 10]. Herein, we recruited an UPLC-QTOF/MS method to profile the effective components of LCD, and the unknown components were classified and assigned based on the fragmentation patterns and diagnostic ions of different structural types of components. According to the component characterization result of LCD in vitro, the prototypes in plasma, urine, and feces were further analyzed based on the similarity of mass spectrometry behavior (accurate molecular weight and secondary fragments) and chromatographic behavior (retention time). Metabolites were matched e mass defect filtering (MDF) caused by biotransformation and were further confirmed by MS/MS spectrum analysis.

2. Material and Methods

2.1. Chemicals and Drugs

LCD was prepared by the Pharmaceutical Department, Shenzhen Traditional Chinese Medicine Hospital. The Chinese medicine including Codonopsis Radix (Lot: 190505101, root of Codonopsis pilosula (Franch.) Nannf.), Atractylodis Macrocephalae Rhizoma (Lot: 1904001, rhizoma of Atractylodes macrocephala Koidz.), Chebulae Fructus (Lot: 181203361, fructus of Terminalia chebula Retz.), Halloysitum Rubrum (Lot: 190300991), Sophorae Flos (Lot: 190504381, flos of Sophora japonica L.), Typhae Pollen (Lot: 190401, pollen of Typha angustifolia L.), Ailanthi Cortex (Lot: 181001, cortex of Ailanthus altissima (Mill.) Swingle), Bletillae Rhizoma (Lot:HX19K01, rhizoma of Bletilla striata (Thunb.) Reichb. f), Coicis Semen (Lot: 1905002, semen of Coix lacryma-jobi L. var. ma-yuen (roman) Stapf), Notoginseng Radix et Rhizoma (Lot: 190401411, radix and rhizoma of Panax notoginseng (Burk.) F. H. Chen), Cynanchi Paniculati Radix et Rhizoma (Lot: 190403711, radix and rhizoma of Cynanchum paniculatum (Bge.) Kitag.), and Glycyrrhizae Radix et Rhizoma (Lot: 1905001, radix and rhizoma of Glycyrrhiza uralensis Fisch.) was purchased from Kangmei Pharmaceutical Co., Ltd (Puning, China). Trigonelline, chebulic acid, gallic acid, 6,7-dihydroxycoumarin, corilagin, typhaneoside, rutin, hyperoside, liquiritin, nicotiflorin, lobetyolin, ginsenoside Re, ginsenoside Rg1, quercetin, ginsenoside Rb1, naringenin, 20S-ginsenoside Rh1, isorhamnetin, ginsenoside Rd, and glycyrrhizic acid, a total of 20 reference standards, were purchased from Chengdu Alfa Biotechnology Co., Ltd. The purity of each compound was more than 98% determined by the HPLC analysis. Methanol was of HPLC grade. Ultrapure water was obtained by the filtration of distilled water using a Milli-Q system (Millipore, USA). LC-MS grade acetonitrile was purchased from Fisher Scientific (Fair Lawn, New Jersey, USA), and LC-MS grade formic acid was purchased from Sigma-Aldrich (St, Missouri, USA).

2.2. Animal

Male Sprague-Dawley rats (300 ± 20 g) were obtained from the Medical Experimental Animal Center of Guangzhou University of Chinese Medicine, China. Rats were housed in specified pathogen-free conditions (23 ± 2°C) under a 12-h light/12-h dark cycle and given free access to food and water. The protocols were approved by the Animal Experimental Ethics Committee of Guangzhou University of Chinese Medicine (Guangzhou, China).

2.3. LCD Preparation

The Medicine Codonopsis Radix, Atractylodis Macrocephalae Rhizoma, Chebulae Fructus, Halloysitum Rubrum, Sophorae Flos, Typhae Pollen, Ailanthi Cortex, Bletillae Rhizoma, Coicis Semen, Notoginseng Radix et Rhizoma, Cynanchi Paniculati Radix et Rhizoma, and Glycyrrhizae Radix et Rhizoma were weighed and mixed at a ratio of 6 : 3:3 : 6:3 : 3:6 : 3:6 : 2:6 : 2. The total weight of LCD is 245g, and the mixture was extracted twice by boiling in distilled water, and eight times distilled water (1960 ml) (w/v) was used to boil for 40 min in the first time, which changes to four times distilled water (980 ml) (w/v) in the second time. The two extracts were merged and centrifuged at 3,000 rpm, for 5 min to exclude dregs, and the supernatant was concentrated to 3.185 g/ml under reduced pressure at 55°C.

2.4. Rat Treatment and Sample Collection

The dose of LCD used in this experiment is 22.05 g/kg, which is the biological equivalent dose of humans. Three rats were fasted for 12 h with free access to drinking water, and then, the rats were orally administered with LCD. LCD was diluted to 2.205 g/ml with distilled water before giving to rat. Then, the blood samples were collected in the heparin anticoagulant tube through retro-orbital plexus at 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 12 h. The plasma samples were obtained by centrifugation at 3000 rpm for 10 min. Samples of the same point were combined and stored at −80°C until use. Feces and urine samples were collected during 0–12 h.

2.5. Biological Sample Preparation

For the plasma sample, about 200 μl plasma was mixed with 600 μl acetonitrile (containing 0.2% methanoic acid). After vortexing for 2 min, the samples were centrifuged at 13000 rpm, 4°C, 10 min. Then, 400 μl supernatant was removed, dried under nitrogen gas, and redissolved in 100 μl acetonitrile (50%). Finally, the samples were centrifuged at 13000 rpm, 4°C, 10 min, and a 2 μl aliquot was injected into UPLC-QTOF-MS. For the fecal sample, about 300 mg of feces was weighed and mixed with 1 ml methanol. After the addition of magnetic beads, the samples were homogenized using tissue grinders (Shanghai Jingxin, Shanghai, China) and centrifuged at 13000 rpm, 4°C, 10 min. About 200 μl supernatant was removed, dried under nitrogen gas, and redissolved in 200 μl acetonitrile (50%). Finally, the samples were centrifuged at 13000 rpm, 4°C, 10 min, and a 2 μl aliquot was injected into UPLC-QTOF-MS. For the urine sample, the mixed urine was centrifuged at 4000 rpm for 10 min, and 1 ml supernatant was loaded on pre-activated Sep-Pak Vac C18 columns (3 cc, 500 mg, Waters, Ireland). After washing with 1 ml ultrapure water and eluting with 1 ml methanol, the elution was collected and centrifuged at 13000 rpm, 4°C, 10 min. About 400 μl supernatant was transferred and dried under nitrogen gas. The residues were redissolved in 400 μl acetonitrile (50%). Finally, the samples were centrifuged at 13000 rpm, 4°C, 10 min, and a 2 μl aliquot was injected into UPLC-QTOF-MS.

2.6. UPLC-QTOF-MS Analysis Condition

The separation equipment for this assay was Sciex Exion LC, and the chromatographic column was Waters Acquity HSS T3 (2.1 × 150 mm, 1.7 μm). The temperature was set at 35°C, and the flow rate was 0.3 ml/min. The mobile phases were 0.1% formic acid in water (A) and acetonitrile (B), with the optimized gradient as follows: 0–5 min from 3% B to 8% B, 5–11 min from 8% B to 30% B, 11–20 min from 30% B to 80% B, 20–21 min from 80% B to 95% B, 21–25 min was maintained at 95% B, and then back to the initial ratio and re-equilibration for 7 min. The 5600 QTOF mass spectrometer (AB Sciex, Foster City, CA, USA) equipped with an ESI ion source was operated in positive and negative modes, and the mass range was m/z of 100–1250. The details of mass spectrometry conditions were summarized as follows: gas 1 and gas 2, 45 psi; curtain gas, 35 psi; heat block temperature, 500°C; ion spray voltage, −4.5 kV in negative mode and 5.5 kV in positive; declustering potential, 50V; collision energy, ±35 V; and the collision energy spread (CES), ±15 V. Sciex OS 1.6.1 was the basal data processing platform, and MetabolitePilot 2.0.4 software was applied for further metabolite fishing.

3. Results and Discussion

3.1. Characterization of Chemical Compounds in LCD

The base peak chromatograms of LCD in negative and positive ion modes are shown in Figure 2. A total of 104 chemical components, including 20 saponins, 26 flavonoids, 5 tannins, 20 organic acids, 8 amino acids, 2 alkaloids, 5 oligosaccharides, and 3 lignans, were identified or tentatively characterized by UPLC-QTOF-MS. As the result of chemical composition classification is summarized in Table 1, CR mainly contained alkaloid compounds and oligosaccharides, while NRR was characterized by saponins. Besides, the major constituents of SF were flavonoids. GRR contains saponins and flavonoids, and CPRR was as characterized by the C21 type steroidal saponins. The characteristic ingredients of TP were flavonoids and organic acids. CF was characterized by the component of tannins; AMR contains organic acids and esters.
Figure 2

Base peak chromatogram (BPC) of LCD.

Table 1

Chemical component of LCD.

AlkaloidAmino acidOligosaccharidesSaponinsLignansFlavonoidsOrganic acidsTanninsOthers (Nucleosides, glycosides, esters, etc.)Total
CR245—————6 (3)17
NRR———9—————9
BR————3—1—15
SF—1—2—8 (3)——213
GRR———8—14——123
CPRR———1—————1
TP—3———5 (2)6 (2)—2 (1)16
CF——————5 (1)5111
AMR——————1—2 (1)3
CS—————3 (3)10 (4)—2 (1)15
AC—————2 (2)1 (1)—14
Total2852032620515104

The number in the brackets was the repeat compounds.

Generally, the characteristic components of AC were triterpenes, and the CS was characterized by lipids. However, both chemical categories were difficult to extract by water so that only flavonoids and organic acids in AC and CS were still detected and identified. Figure 3 draws the part of representative structures of each medicine.
Figure 3

Representative structures of each medicine of LCD.

3.2. Fragmentation Mechanisms of Medicine Representative Structures

3.2.1. Codonopsis Radix-Derived Compounds

A total of 17 compounds were identified in CR. Among them, saccharides (P4 fructose, P6 sucrose, P7 raffinose, P8 stachyose, and P14 verbascose) and alkaloids (P5 trigonelline and P25 codonopsine) were characteristic components [11, 12]. Saccharides showed [M-H]− in the negative ion mode and [M + NH4]+/[M + Na]+in the positive ion mode. The successive neutral loss of hexose (−162 Da) and H2O (−18 Da) was used for identification. The typical fragmentation pattern of P14 verbascose is drawn in Figure 4(a). Alkaloid P5 trigonelline produced a [M + H]+ ion at m/z of 138.0546 and had fragment ions at m/z of 94, 92, and 78, which correspond to [M - CO2 + H]+, C6H6N+, and C5H4N+, respectively. P25 codonopsine showed [M + H]+ ion at m/z of 268.1546, and the fragment ions at m/z of 161, 88, and 58 were produced by penta-heterocycle cracking. The typical fragmentation pathways of P5 trigonelline are drawn in Figure 4(b).
Figure 4

MS/MS spectrum and major fragmentation pathways of representative structure in LCD. (a) P14 verbascose; (b) P5 trigonelline; (c) P59 ginsenoside Rg1; (d) P47 dactylorhin A; (e) P43 rutin; (f) P89 paniculatumoside A; (g) P36 corilagin; (h) P92 atractylenolide III; (i) P104 20-R-hydroxydammara-24-en-3-one.

3.2.2. Notoginseng Radix et Rhizoma-Derived Compounds

About 9 compounds were identified in Notoginseng Radix et Rhizoma, and all of the compounds were triterpenoid saponins (P55 notoginsenoside E, P58 ginsenoside Re, P59 ginsenoside Rg1, P72 ginsenoside Rb1, P74 notoginsenoside R2, P76 20s-ginsenoside Rh1, P77 ginsenoside Rh4/Rk3, P82 ginsenoside Rd, and P91 ginsenoside F2) [13-15]. The neutral loss of Glc (162 Da) and Rha (146 Da) was characteristically appeared in saponin compounds. P59 ginsenoside Rg1 is taken as example, and it had the [M + HCOO]− ion at m/z of 845.4899 and [M + H]+ ion at m/z of 801.4983. The characteristic product ions at m/z of 621 [M-Glc-H2O]+, 603 [M-Glc-2H2O]+, 441 [M-2Glc-2H2O]+, 423 [M-2Glc-3H2O]+, and 405 [M-2Glc-4H2O]+ were observed. The typical fragmentation pathways of P59 ginsenoside Rg1 are drawn in Figure 4(c).

3.2.3. Bletillae Rhizoma-Derived Compounds

A total of 5 characteristic compounds were detected in Bletillae Rhizoma. P47 dactylorhin A [16], P56 gymnoside III, and P61 militarine [17] were structurally similar to that contained two molecules of gastrodin (P22). Neutral loss of Glc (162 Da), H2O (18 Da), and gastrodin (268 Da) was used for identification. P47 dactylorhin A showed the [M - H]− ion at m/z of 887.3181 and [M + NH4]+ ion at m/z of 906.3601, while it had characteristic fragment ion at [M-Glc-H2O–H]− at m/z of 707, [M-gastrodin-H]− at m/z of 619, [M-gastrodin-Glc- H2O–H]− at m/z of 439, [M-gastrodin-Glc + H]+ at m/z of 459, [gastrodin]+ at m/z of 269, and [gastrodin-Glc]+ at m/z of 107. The typical fragmentation pathways of P47 dactylorhin A are drawn in Figure 4(d).

3.2.4. Sophorae Flos-Derived Compounds

Thirteen compounds were isolated from Sophorae Flos [18-20], and more than half of them were flavonoids, or specifically flavonols (P37 quercetin 3-O-glucosyl-rutinoside [21], P39 manghaslin [22], P43 rutin [23-25], P45 isoquercitrin [26], P48 nicotiflorin [27], P50 narcissin [24, 28], P70 quercetin [23, 29, 30], and P79 isorhamnetin [22]). In negative mode, flavonoid glycosides were trend to neutral loss of glycosides. In addition, neutral losses of CH3 (15 Da), CO (28 Da), and RDA cracking could also be observed. P43 rutin was a vital constituent of Sophorae Flos. It had the [M + H]+ ion at m/z of 611.1607 and gave characteristic fragment ions at m/z of 465 and 303 by successive loss of Glc (162 Da) and Rha (146 Da). The typical fragmentation pathways of P43 rutin are drawn in Figure 4(e).

3.2.5. Glycyrrhizae Radix et Rhizoma-Derived Compounds

A total of 23 compounds were discriminated in Glycyrrhizae Radix et Rhizoma, and 14 of them were flavonoids (P44 licuraside/liquiritin apioside, P46 liquiritin, P54 naringenin-7-O-glucoside, P60 violanthin, P67 pallidiflorin, P69 isoliquiritigenin [31-33], P63 licorice glycoside B/D1, P64 licorice glycoside C2, P66 licorice glycoside E, P75 naringenin [34], P53 choerospodin [35], P62 ononin/ononin isomer [36], P90 glyasperin C [37],and P93 sophoraisoflavone A/semilicoisoflavone B [38]). Different from sophorae, the flavonoids in glycyrrhiza were more abundant, including chalcone, flavones, and flavanones. However, the primary cracking patterns such as neutral loss of glycosides were similar. In addition to flavonoids, triterpenoid saponins were characteristic components as well. Representative compound licorice saponin A3 (P73, [M - H]− at m/z of 983.4455, [M + H]+ at m/z of 985.4644) observed fragments ions at [M-GlcA + H]+ at m/z of 809, [M-Glc-GlcA + H]+ at m/z of 647, [M-2GlcA-H2O + H]+ at m/z of 615, [M-Glc-2GlcA + H]+ at m/z of 471, and [M-Glc-2GlcA-H2O + H]+ at m/z of 453. The fragmentation pathways were similar to P59 drawn in Figure 4(c).

3.2.6. Cynanchi Paniculati Radix et Rhizoma-Derived Compounds

Only one special saponin (steroidal glycoside), namely paniculatumoside A or B (P89) [39], was identified in Cynanchi Paniculati Radix et Rhizoma. The cracking mainly occurred at A (m/z of 331) and A' rings (m/z of 145, 113). The typical fragmentation pathways of P89 are drawn in Figure 4(f).

3.2.7. Typhae Pollen-Derived Compounds

In this experiment, the characteristic components detected in Typhae Pollen were flavonoids (P42 typhaneoside [40], P43 rutin [23-25], P49 isorhamnetin-3-O-rutinoside-7-O-rhamnoside [24], P50 narcissin [24, 28], and P52 isorhamnetin-3-O-beta-galactoside [40]) and carboxylic acids (P9 L-malic acid [23, 40], P10 citric acid [40], P18 succinic acid [40], P27 3,4-dihydroxybenzoic acid, P51 vanillic acid [23], and P68 decanedioic acid [41]). Typhaneoside (P42), [M-H]− at m/z of 769.2194, [M +H ]+ at m/z of 771.2327) was a flavonol, and fragment ions were observed after successive loss of Rha (146 Da) and Glc (162 Da). The fragmentation pathways were similar to P43 drawn in Figure 4(e). Simple carboxylic acids were generally responded in the negative mode, and neutral loss of·CH3 (15 Da), H2O (-18 Da), and CO2 (-44 Da) was the most usual fragments.

3.2.8. Chebulae Fructus-Derived Compounds

In Chebulae Fructus, gallic acid structure was found in carboxylic acids (P13 chebulic acid [42], P23 gallic acid [23, 43], P26 5-galloylshikimic acid [44], P33 brevifolincarboxylic acid [45], and P40 3,4,8,9,10-pentahydroxydibenzo[b,d]pyran-6-one [44]), while ellagic acid (gallic acid dimer) structure was tannins (P28 hamamelitannin [46], P29 1,6-di-O-galloyl-β-D-glucose [47], P34 chebulanin(1-O-galloyl-2,4-O-chebuloyl-b-D-Glc [44]), P36 corilagin [48], and P41 chebulagic acid [46]). Thus, ellagic acid fragment (m/z of 300) and neutral loss of gallic acid (170 Da) could be generally observed. The typical fragmentation pathways of P36 corilagin are drawn in Figure 4(g).

3.2.9. Atractylodis Macrocephalae Rhizoma-Derived Compounds

The characteristic compound in Atractylodis Macrocephalae Rhizoma was lactone (P92 atractylenolide III [11, 49]). Lactone was generally responded in the positive mode. Atractylenolide III (P92, [M + H]+ at m/z of 249.1487) showed fragment ions at [M-H2O + H]+ at m/z of 231, [M-H2CO2+H]+ at m/z of 185, [M-C3H4O + H]+ at m/z of 175, C10H10O2+ at m/z of 163, and C6H7+ at m/z of 79. The typical fragmentation pathways of P92 atractylenolide III are drawn in Figure 4(h).

3.2.10. Coicis Semen-Derived Compounds

A total of 15 compounds could be attributed to coicis semen, including 10 carboxylic acid (P9 L-malic acid [23, 40], P23 gallic acid [23, 43], P51 vanillic acid [23], P95 pseudolaroside B, P96 quinic acid [23], P97 protocatechuic acid [50], P98 caffeic acid [50], P99 nonanedioic acid [51], P100 1-caffeoylquinic acid [52], and P101 3-O-feruloylquinic acid [52]), 3 flavonoids (P43 rutin [23-25], P70 quercetin [23, 29, 30],and P103 kaempferol [29, 50]), 1 phenylpropanoid (P19 p-coumaric acid [11, 53]), and 1 nucleoside (P102 adenosine [53]).

3.2.11. Ailanthi Cortex-Derived Compounds

In Ailanthi Cortex, 4 compounds were attributed: briefly, 2 flavonoids (P70 quercetin [23, 29, 30] and P103 kaempferol [29, 50]), 1 carboxylic acid (P99 nonanedioic acid [51]), and 1 terpene (P104 20-R-hydroxydammara-24-en-3-one). However, only P104 was characteristic, and it had the [M + H]+ ion at m/z of 443.3881 and gave fragment ions at m/z of 425 by neutral loss of H2O (18 Da). The crack of C ring formed ions at m/z of 221 and 207. The typical fragmentation pathways of P104 are drawn in Figure 4(i).

3.3. Characterization of LCD-Related Xenobiotics in Rat Biological Samples

According to the compound characterization of LCD, the fragmentation patterns of mass spectrometry (accurate molecular weight and secondary debris) and retention time of chromatography were adopted to analyze the components in plasma, urine, and feces. P59 ginsenoside Rg1 is taken as example, as shown in the XIC of LCD (Figure 5(a)) and multiple XICs of 6 bio-samples (Figure 5(b)), and a peak at 13.4 min was clearly observed in administration of bio-samples but not in the blanks. Importantly, the MS/MS spectra (m/z of 621, 441, 423, 405, and 203) of ginsenoside Rg1 in LCD (Figure 5(c)) and bio-samples (Figure 5(d)) were similar.
Figure 5

Identification of prototypes in bio-samples, and P59 ginsenoside Rg1 is taken as an example. (a) XIC of ginsenoside Rg1 in LCD; (b) multiple XICs of ginsenoside Rg1 in bio-samples. From top to bottom: administration plasma, blank plasma, administration urine, blank urine, administration feces, and blank feces. Ginsenoside Rg1 showed the highest intensity in feces, lowest in plasma, and no response in blank samples; (c) MS/MS spectrum of ginsenoside Rg1 in LCD; (d) MS/MS spectrum of ginsenoside Rg1 in feces.

Based on the above principles, a total of 50 components were matched in biological samples, and these components would play a key role in explaining the mechanism of LCD in the future. In particular, flavonoids (P43, P46, and P50) and saponins (P55 and P72) deserved higher attention as the five components were observed in all three bio-samples besides that were common to organisms (P1, P11, P15, P24, P31, and P68). In addition, 12 compounds were just observed in the fecal sample, mainly including some alkaloids (P25 and P65), flavonoids (P37, P42, P45, P52, and P70), saponin (P74), and other small molecules (P6, P9, P35, and P40). These compounds may not be absorbed into the blood, but are still effective in regulating gut microbiota. The detailed information about the distribution of components in plasma, urine, and feces is summarized in Table 2.
Table 2

Identification of the major components present in LCD by UPLC-QTOF-MS.

No.CompoundFormulaRt (min)Ion modeCal m/zESI-m/zppmFragment ions (m/z)Ion modeCal m/zESI+m/zppmFragment ions (m/z)Compound classSourceReference
P1CholineC5H13NO1.25—————[M + H]+104.1070104.1065−4.860, 59CholineCR[11]
P2ArginineC6H14N4O21.21[M - H]−173.1039173.10400.6173.131[M + H]+175.1190175.1189−0.6130, 116, 70, 60Amino acidCR[11]
P3AsparagineC4H8N2O31.24[M - H]−131.0457131.04623.8114, 113, 95, 72.58[M + H]+133.0608133.0606−1.574Amino acidCR[11]
P4FructoseC6H12O61.33[M - H]−179.0556179.0555−0.6161, 131, 101, 85, 59[M + Na]+203.0526203.0524−1.0158.88.70SaccharidesCR[11]
P5Trigonelline∗C7H7NO21.36—————[M + H]+138.0550138.0546−2.994, 92, 78,AlkaloidsCR[11]
P6SucroseC12H22O111.43[M - H]−341.1089341.10890.0179, 89—————SaccharidesCR[11]
P7RaffinoseC18H32O161.51[M - H]−503.1618503.1606−2.4323, 191, 179[M + NH4]+522.2029522.2019−1.9325, 289, 163, 145, 127SaccharidesCR[11]
P8StachyoseC24H42O211.65[M - H]−665.2146665.2133−2.0341, 323, 179, 161[M + NH4]+684.2557684.2549−1.2487, 325, 289, 163, 145, 127SaccharidesCR[11]
P9L-Malic acidC4H6O51.66[M - H]−133.0142133.01420.0115, 89, 71—————Carboxylic acidsTP/CS[23, 40]
P10Citric acidC6H8O71.69[M - H]−191.0197191.02001.6111, 85, 73—————Carboxylic acidsTP[40]
P11ValineC5H11NO21.69—————[M + H]+118.0863118.0854−7.672, 55Amino acidTP[41]
P12Adenine nucleosideC10H13N5O41.76/3.20—————[M + H]+268.1040268.1038−0.7136, 119NucleosideCR[11]
P13Chebulic acid∗C14H12O111.80/2.27[M - H]−355.0307355.0296−3.1337, 293, 249, 205—————Carboxylic acidsCF[42]
P14VerbascoseC30H52O262.00[M - H]−827.2669827.26740.6665, 503, 341, 179, 161[M + Na]+851.2639851.2618−2.5689SaccharidesCR[11]
P15IsoleucineC6H13NO22.07—————[M + H]+132.1019132.1013−4.586, 85Amino acidCR[11]
P16L-Pyroglutamic acidC5H7NO32.41[M - H]−128.0348128.03533.982[M + H]+130.0499130.0493−4.684.56Amino acidCR[11]
P17UridineC9H12N2O62.66[M - H]−243.0623243.06230.0200, 152, 110[M + H]+245.0768245.07700.8113, 70NucleosideTP[12]
P18Succinic acidC4H6O42.70[M - H]−117.0193117.0192−0.973—————Carboxylic acidsTP[40]
P19p-Coumaric acidC9H8O32.86—————[M + H]+165.0546165.0541−3.0162.123.77PhenylpropanoidsCR/CS[11, 53]
P20LeucineC6H13NO23.10—————[M + H]+132.1019132.1014−3.886Amino acidTP[41]
P21GuanosineC10H13N5O53.74[M - H]−282.0838282.08411.1150, 133, 107—————NucleosideCR/TP[11, 12]
P22GastrodinC13H18O73.85—————[M + NH4]+304.1391304.13961.6108, 107, 105GlycosideBR[17]
P23Gallic acid∗C7H6O54.17[M - H]−169.0142169.01462.4125[M + H]+171.0288171.0281−4.1153, 107Carboxylic acidsCF/CS[23, 43]
P24PhenylalanineC9H11NO25.12[M - H]−164.0717164.07180.6147, 103, 72[M + H]+166.0863166.0859−2.4120, 103, 77Amino acidTP[41]
P25CodonopsineC14H21NO46.27—————[M + H]+268.1543268.15461.1161, 121, 88, 58AlkaloidsCR[11]
P265-Galloylshikimic acidC14H14O96.74[M - H]−325.0565325.05701.5169, 125—————Carboxylic acidsCF[44]
P273, 4-Dihydroxybenzoic acidC7H6O46.93[M - H]−153.0193153.01972.6109, 108—————Carboxylic acidsTP—
P28HamamelitanninC20H20O147.95[M - H]−483.0780483.0773−1.4271, 211, 169, 125—————TanninsCF[46]
P291, 6-Di-O-galloyl-β-D-glucoseC20H20O148.52/8.91/9.09/9.25[M - H]−483.0780483.0779−0.2423, 271, 211, 169—————TanninsCF[47]
P305-Hydroxyferulic acidC10H10O58.99[M - H]−209.0456209.04612.4165, 121, 59—————Carboxylic acidsAMR-
P314-Hydroxybenzoic acidC7H6O39.16[M - H]−137.0244137.0241−2.293—————Carboxylic acidsBR[16]
P32Soyamaloside CC23H32O169.61[M - H]−563.1618563.1614−0.7461, 419—————GlycosideSF[19]
P33Brevifolincarboxylic acidC13H8O89.73[M - H]−291.0141291.01472.1247, 219, 191—————Carboxylic acidsCF[45]
P34Chebulanin(1-O-galloyl-2, 4-O-chebuloyl-b-D-Glc)C27H24O199.98[M - H]−651.0834651.08390.8633, 481, 275, 169—————TanninsCF[44]
P356, 7-Dihydroxycoumarin∗C9H6O410.04[M - H]−177.0188177.01922.3177, 133, 105, 89—————CoumarinsGRR[34]
P36Corilagin∗C27H22O1810.18[M - H]−633.0704633.07324.4463, 300, 169[M + NH4]+652.1145652.1140−0.7465, 363, 303, 277TanninsCF[48]
P37Quercetin 3-O-glucosyl-rutinosideC33H40O2110.37[M - H]−771.1993771.1990−0.4300[M + H]+773.2136773.2134−0.3465, 303FlavonoidsSF[21]
P38Euphormisin M3C27H24O1810.55[M - H]−635.0890635.08910.2483, 465, 169, 125[M + NH4]+654.1301654.1280−3.3467, 297, 171, 153GlycosideCF[46]
P39ManghaslinC33H40O2010.58[M - H]−755.2040755.20410.1609, 447, 299[M + H]+757.2187757.2179−1.0661, 449, 303FlavonoidsSF[22]
P403,4,8,9,10-Pentahydroxydibenzo[b,d]pyran-6-oneC13H8O710.96[M - H]−275.0192275.02013.3258, 257, 229, 201, 173, 145,—————Carboxylic acidsCF[44]
P41Chebulagic acidC41H30O2711.05[M - H]−953.0896953.09030.7301, 275—————TanninsCF[46]
P42Typhaneoside∗C34H42O2011.25[M - H]−769.2197769.2194−0.4623, 314, 189[M + H]+771.2343771.2327−2.1625, 479, 317FlavonoidsTP[40]
P43Rutin∗C27H30O1611.56[M - H]−609.1461609.1459−0.3301[M + H]+611.1607611.1607−0.1465, 303, 85, 71FlavonoidsSF/TP/CS[23–25]
P44Licuraside/liquiritin apiosideC26H30O1311.65[M - H]−549.1608549.16110.5255,135[M + H]+551.1765551.1741−4.4257,137FlavonoidsGRR[31]
P45Hyperoside∗C21H20O1211.87[M - H]−463.0882463.0869−2.8300, 301[M+H]+465.1028465.1021−1.5303FlavonoidsSF[26]
P46Liquiritin∗C21H22O911.85[M - H]−417.1191417.1184−1.7255, 135[M + NH4]+436.1603436.1592−2.5257, 137FlavonoidsGRR[31]
P47Dactylorhin AC40H56O2212.06[M - H]−887.3190887.3181−1.0707, 619, 439[M + NH4]+906.3603906.3601−0.3621, 537, 459, 431, 403, 375, 325, 297, 269, 213, 191, 107LignansBR[16]
P48Nicotiflorin∗C27H30O1512.18[M - H]−593.1512593.1502−1.7285[M + H]+595.1658595.1657−0.2449, 431, 287FlavonoidsSF[27]
P49Isorhamnetin-3-O-rutinoside-7-O-rhamnosideC34H40O2012.23[M - H]−767.2040767.20460.8705, 665, 623, 314, 299, 271, 179—————FlavonoidsTP[24]
P50NarcissinC28H32O1612.27[M - H]−623.1618623.1600−2.9315, 314, 300, 285, 271, 255, 151[M + H]+625.1763625.1754−1.4317FlavonoidsTP/SF[24, 28]
P51Vanillic acidC8H8O412.60[M - H]−167.0350167.0347−1.8152,108—————Carboxylic acidsTP/CS[23]
P52Isorhamnetin-3-O-beta-galactosideC22H22O1212.70[M - H]−477.1038477.1027−2.3314, 285[M + H]+479.1184479.1168−3.3317FlavonoidsTP[40]
P53ChoerospodinC21H22O1012.83[M - H]−433.1140433.11512.5271, 151[M + H]+435.1286435.1268−4.1273, 153FlavonoidsGRR[35]
P54Naringenin-7-O-glucosideC21H22O1012.95[M - H]−433.1140433.11512.5433, 271, 151[M + H]+435.1286435.1274−2.8153,147FlavonoidsGRR[31]
P55Notoginsenoside EC48H82O2012.96[M - H]−977.5321977.5308−1.3931—————SaponinsNRR[13]
P56Gymnoside IIIC42H58O2313.22[M - H]−975.3351975.3336−1.5707, 661, 439[M + NH4]+948.3709948.3692−1.8825, 663, 635, 501, 473, 395, 367, 297, 205, 107LignansBR—
P57Lobetyolin∗C20H28O813.24—————[M + NH4]+414.2124414.2121−0.6199, 155GlycosideCR[11]
P58Ginsenoside Re∗C48H82O1813.34/15.12/16.09/16.51[M + COOH]−991.5483991.5459−2.4783, 621[M + H]+947.5577947.5544−3.5767, 749, 605, 587, 443, 407, 325SaponinsNRR[14]
P59Ginsenoside Rg1∗C42H72O1413.40[M + COOH]−845.4904845.4899−0.6799, 637[M + H]+801.4998801.4983−1.8621, 603, 441, 423, 405, 325SaponinsNRR[14]
P60ViolanthinC27H30O1413.65[M - H]−577.1563577.1553−1.7515, 475, 433, 145[M+H]+579.1708579.1701−1.2453, 291, 147FlavonoidsGRR[31]
P61MilitarineC34H46O1713.65[M + COOH]−771.2717771.2702−1.9725, 457, 285, 153[M + NH4]+744.3075744.3069−0.8107LignansBR[17]
P62Ononin/ononin isomerC22H22O913.73—————[M + H]+431.1342431.1337−1.2269FlavonoidsGRR[36]
P63Licorice glycoside B/D1C35H36O1513.73[M - H]−695.1981695.1961−2.9255, 399, 531, 549—————FlavonoidsGRR[34]
P64Licorice glycoside C2C36H38O1613.81[M - H]−725.2087725.2076−1.5549, 531, 255, 193[M + H]+727.2233727.22330.0309,297,245FlavonoidsGRR[34]
P65N, N′-diferuloylputrescineC24H28N2O614.17[M - H]−439.1875439.18852.3289, 149[M + H]+441.2020441.2009−2.5265, 177Amino acidSF[18]
P66Licorice glycoside EC35H35NO1414.34[M - H]−692.1985692.1983−0.3549, 531[M + H]+694.2130694.2114−2.3240, 144FlavonoidsGRR[34]
P67PallidiflorinC16H12O414.42[M - H]−267.0663267.0661−0.7267, 252, 195, 132—————FlavonoidsGRR[31]
P68Decanedioic acidC10H18O414.45[M - H]−201.1132201.1125−3.5183, 139,—————Carboxylic acidsTP[41]
P69IsoliquiritigeninC15H12O414.46[M - H]−255.0663255.0655−3.1255, 135, 119, 91[M + H]+257.0808257.08163.1257, 147, 137, 119, 81FlavonoidsGRR[31]
P70Quercetin∗C15H10O714.67[M - H]−301.0354301.0346−2.7179, 151[M + H]+303.0499303.05031.3245, 301, 106, 151FlavonoidsSF/CS/AC[23, 29, 30]
P71Licorice saponin A3C48H72O2114.69[M - H]−983.4493983.4455−3.9821, 645, 351[M + H]+985.4642985.46440.3809, 647, 615, 471, 453SaponinsGRR[31]
P72Ginsenoside Rb1∗C54H92O2315.13[M + HCOOH–2H]2-599.2997599.2987−1.71107, 945, 783, 553, 161[M + H]+1109.61061109.6078−2.5767, 649, 605, 487, 425, 407, 325, 289SaponinsNRR[14]
P73Licorice saponin G2C42H62O1715.24[M - H]−837.3914837.3898−1.9351[M + H]+839.4062839.4046−1.9839, 663, 487, 469SaponinsGRR[31]
P74Notoginsenoside R2C41H70O1315.31[M + COOH]−815.4799815.4787−1.5769, 637—————SaponinsNRR[13]
P75Naringenin∗C15H12O515.57[M - H]−271.0604271.06123.0151, 119[M + H]+273.0757273.07601.1153, 147FlavonoidsGRR[34]
P7620S-Ginsenoside Rh1∗C36H62O915.71[M + COOH]−683.4376683.4359−2.5673, 475—————SaponinsNRR[15]
P77Ginsenoside Rh4/Rk3C36H60O815.76—————[M + H]+621.4364621.4361−0.4441, 423, 405, 221, 203, 187SaponinsNRR[15]
P78Licorice saponin G2 isomerC42H62O1715.83[M - H]−837.3914837.3901−1.6351[M + H]+839.4062839.40650.3839, 663, 645, 487, 469SaponinsGRR[31]
P79Isorhamnetin∗C16H12O715.95—————[M + H]+317.0656317.06590.9302, 153FlavonoidsSF[22]
P80Raho glycyrrhizinC48H72O2015.96[M - H]−967.4544967.4517−2.8329[M + H]+969.4692969.4650−4.4621, 453, 435, 405, 217SaponinsGRR[32]
P81BetulinC30H50O216.10[M - H]−————[M + H]+443.3884443.38860.5443, 425, 407, 271, 207, 175, 59TriterpenoidsSF[20]
P82Ginsenoside Rd∗C48H82O1816.11[M + COOH]−991.5483991.5459−2.4783, 621—————SaponinsNRR[13]
P83Yunganoside G1C48H74O2116.14—————[M + H]+987.4798987.4779−1.9841, 665, 629, 471, 453, 441, 353SaponinsGRR[33]
P84Glycyrrhizic acid∗C42H62O1616.31[M - H]−821.3965821.3942−2.8759, 351, 193[M + H]+823.4113823.4111−0.2823, 647, 471, 453, 194SaponinsGRR[31]
P85Glycyrrhizic isomer /uralsaponin A/licorice saponin K2/licorice saponin H2C42H62O1616.82/17.02[M - H]−821.3965821.3953−1.5351, 193[M + H]+823.4113823.4111−0.2823, 647, 471, 453, 194SaponinsGRR[31]
P86Kaikasaponin IIIC48H78O1717.15[M + COOH]−971.5221971.5194−2.8925[M + NH4]+944.5580944.5553−2.9503, 485, 425, 407, 309, 287, 147SaponinsSF[19]
P87Uralsaponin C/licorice saponin J2C42H64O1617.22—————[M + H]+825.4270825.4248−2.6825, 613, 455, 409, 397, 317, 177, 159, 141SaponinsGRR[31]
P88Kaikasaponin IC42H68O1317.73—————[M + NH4]+798.5001798.4988−1.6425, 407, 339, 163SaponinsSF[19]
P89Paniculatumoside A/paniculatumoside BC28H40O918.00—————[M + H]+521.2747521.2739−1.5331, 145, 113Saponins (steroidal glycoside)CPRR[39]
P90Glyasperin CC21H24O518.09—————[M + H]+357.1697357.1693−1.1283, 165, 137, 123FlavonoidsGRR[37]
P91Ginsenoside F2C42H72O1318.57[M - H]−779.4587779.4575−1.5799[M + Na]+807.4868807.4835−4.1785, 767, 443, 407, 325SaponinsNRR[15]
P92Atractylenolide IIIC15H20O318.62—————[M + H]+249.1497249.1487−4.0231, 175, 163, 185, 161, 105, 79LactoneCR/AMR[11, 49]
P93Sophoraisoflavone A/semilicoisoflavone BC20H16O619.91—————[M + H]+353.1020353.1018−0.6335, 311, 299, 215, 199, 153FlavonoidsGRR[38]
P947-[4-(11-hydroxy-undecyloxy)-phenyl]-7-pyridin-3-yl-hept-6-enoic acid ethyl esterC31H45NO420.82—————[M + H]+496.3421496.3392−5.8478, 184, 104EstersAMR[49]
P95Pseudolaroside BC14H18O96.73[M - H]−329.08781329.08831.49163—————Carboxylic acidsCS—
P96Quinic acidC7H12O61.42[M - H]−191.05611191.0561−0.05191—————Carboxylic acidsCS[23]
P97Protocatechuic acidC7H6O46.93[M - H]−153.01933153.019360.20109, 91—————Carboxylic acidsCS[50]
P98Caffeic acidC9H8O410.13[M - H]−179.03498179.0349−0.45135—————Carboxylic acidsCS[50]
P99Nonanedioic acidC9H16O413.2[M - H]−187.09758187.09781.18187, 169, 125, 97, 57[M + H]+189.1121189.11220.53171, 125, 97, 55Carboxylic acidsCS/AC[51]
P1001-Caffeoylquinic acidC17H20O99.72[M - H]−367.10346367.1027−2.07193, 173[M+H]+369.118369.11830.81177, 145Carboxylic acidsCS[52]
P1013-O-Feruloylquinic acidC17H20O910.89[M - H]−367.10346367.1032−0.71193, 191, 173[M + H]+369.118369.11830.81177, 145Carboxylic acidsCS[52]
P102AdenosineC10H13N5O43.35—————[M + H]+268.104268.10420.75136NucleosideCS[53]
P103KaempferolC15H10O615.72—————[M + H]+287.055287.05520.70231, 213, 165, 153, 121FlavonoidsCS/AC[29, 50]
P10420-R-hydroxydammara-24-en-3-oneC30H50O216.08—————[M + H]+443.3884443.3881−0.68425, 221, 207, 189, 133TerpenesAC—

: compounds verified by standards

Furtherly, the phase I and phase II metabolic regularity, as well as the similarity of secondary mass spectrum profile, was used to identify the metabolite. Those metabolites were annotated through automatic matching with prototype components by MetabolitePilot Software. Briefly, MetabolitePilot operated prototype-metabolite matching through mass defect filter (MDF), characteristic product ion filter (PIF), and neutral loss filter (NLF). As shown in Figure 6, the mass defect from P50 to M70/71 was -148 Da with the biotransformation named “loss of C6H10O4 and O (hydrolysis, phase I) + ketone formation (phase I).” Furthermore, neutral loss of glycosides and methylene was both observed in the MS/MS spectra of P50 and M70/71, which implied the similar skeleton. That was to say, these compounds were structurally related, and M70/71 could be the metabolites of P50. As a result, a total of 107 metabolites were matched with 25 prototypes in plasma, urine, or feces. The network of prototype-metabolite matching is drawn as in Figure 7. The details involving the distribution and biotransformations of metabolites are listed in Table 3. It was worth noting that although some prototypes have not been observed in bio-samples, they still are effective through metabolites. For example, P28 hamamelitannin produced 14 metabolites that were all detected in urine, and 5 were found in plasma and 2 in feces. It could be metabolized in the gut, and metabolites were furtherly absorbed into the bloodstream. In total, 29 prototype components and 22 metabolites were detected in plasma. About 27 prototypes and 96 metabolites were detected in urine, and 34 prototypes and 18 metabolites were detected in feces. These substances were considered to constitute the pharmacodynamic substance basis of LCD.
Figure 6

Identification of metabolites in bio-samples.

Figure 7

Correlation between prototype and metabolites.

Table 3

Prototype and metabolic components of LCD in rat serum, urine, and fecal samples.

MetabolitesPrototypeComponent nameFormulatR (min)SerumUrineFeces
—P1CholineC5H13NO1.25√√√
—P2ArginineC6H14N4O21.21√—√
—P3AsparagineC4H8N2O31.24———
—P4FructoseC6H12O61.33———
—P5TrigonellineC7H7NO21.36√√—
—P6SucroseC12H22O111.43——√
—P7RaffinoseC18H32O161.51———
—P8StachyoseC24H42O211.65———
—P9L-Malic acidC4H6O51.66——√
—P10Citric acidC6H8O71.69√√—
—P11ValineC5H11NO21.69√√√
—P12Adenine nucleosideC10H13N5O41.76/3.20√—√
—P13Chebulic acidC14H12O111.80/2.27———
—P14VerbascoseC30H52O262.00———
—P15IsoleucineC6H13NO22.07√√√
—P16L-Pyroglutamic acidC5H7NO32.41√√—
—P17UridineC9H12N2O62.66———
—P18Succinic acidC4H6O42.70√—√
—P19p-Coumaric acidC9H8O32.86√——
—P20LeucineC6H13NO23.10√——
—P21GuanosineC10H13N5O53.74———
—P22GastrodinC13H18O73.85—√—
—P23Gallic acidC7H6O54.17—√√
—P24PhenylalanineC9H11NO25.12√√√
—P25CodonopsineC14H21NO46.27——√
—P265-Galloylshikimic acidC14H14O96.74———
—P273,4-Dihydroxybenzoic acidC7H6O46.93—√√
—P28HamamelitanninC20H20O147.95———
—P291,6-Di-O-galloyl-β-D-glucoseC20H20O148.52/8.91/9.09/9.25———
—P305-Hydroxyferulic acidC10H10O58.99√√—
—P314-Hydroxybenzoic acidC7H6O39.16√√√
—P32Soyamaloside CC23H32O169.61√——
—P33Brevifolincarboxylic acidC13H8O89.73———
—P34Chebulanin(1-O-galloyl-2,4-O-chebuloyl-b-D-Glc)C27H24O199.98———
—P356,7-DihydroxycoumarinC9H6O410.04——√
—P36CorilaginC27H22O1810.18———
—P37Quercetin 3-O-glucosyl-rutinosideC33H40O2110.37——√
—P38Euphormisin M3C27H24O1810.55———
—P39ManghaslinC33H40O2010.58———
—P403,4,8,9,10-Pentahydroxydibenzo[b,d]pyran-6-oneC13H8O710.96——√
—P41Chebulagic acidC41H30O2711.05———
—P42TyphaneosideC34H42O2011.25——√
—P43RutinC27H30O1611.56√√√
—P44Licuraside/liquiritin apiosideC26H30O1311.65—√√
—P45HyperosideC21H20O1211.87——√
—P46LiquiritinC21H22O911.85√√√
—P47Dactylorhin AC40H56O2212.06√√—
—P48NicotiflorinC27H30O1512.18√—√
—P49Isorhamnetin-3-O-rutinoside-7-O-rhamnosideC34H40O2012.23———
—P50NarcissinC28H32O1612.27√√√
—P51Vanillic acidC8H8O412.60√√—
—P52Isorhamnetin-3-O-beta-galactosideC22H22O1212.70——√
—P53ChoerospodinC21H2201012.83———
—P54Naringenin-7-O-glucosideC21H22O1012.95———
—P55Notoginsenoside EC48H82O2012.96√√√
—P56Gymnoside IIIC42H58O2313.22———
—P57LobetyolinC20H28O813.24—√—
—P58Ginsenoside ReC48H82O1813.34√——
—P59Ginsenoside Rg1C42H72O1413.40√—√
—P60ViolanthinC27H30O1413.65—√√
—P61MilitarineC34H46O1713.65√——
—P62Ononin/Ononin isomerC22H22O913.73———
—P63Licorice glycoside B/D1C35H36O1513.73———
—P64Licorice glycoside C2C36H38O1613.81———
—P65N, N′-diferuloylputrescineC24H28N2O614.17——√
—P66Licorice glycoside EC35H35NO1414.34———
—P67PallidiflorinC16H12O414.42—√—
—P68Decanedioic acidC10H18O414.45√√√
—P69IsoliquiritigeninC15H12O414.46—√√
—P70QuercetinC15H10O714.67——√
—P71Licorice saponin A3C48H72O2114.69√√—
—P72Ginsenoside Rb1C54H92O2315.13√√√
—P73Licorice saponin G2C42H62O1715.24—√—
—P74Notoginsenoside R2C41H70O1315.31——√
—P75NaringeninC15H12O515.57———
—P7620S-Ginsenoside Rh1C36H62O915.71———
—P77Ginsenoside Rh4/Rk3C36H60O815.76———
—P78Licorice saponin G2 isomerC42H62O1715.83———
—P79IsorhamnetinC16H12O715.95———
—P80Raho glycyrrhizinC48H72O2015.96———
—P81BetulinC30H50O216.10———
—P82Ginsenoside RdC51H84O2116.11———
—P83Yunganoside G1C48H74O2116.14———
—P84Glycyrrhizic acidC42H62O1616.31———
—P85Glycyrrhizic isomer /uralsaponin A/licorice saponin K2/licorice saponin H2C42H62O1616.82/17.02———
—P86Kaikasaponin IIIC48H78O1717.15———
—P87Uralsaponin C/licorice saponin J2C42H64O1617.22———
—P88Kaikasaponin IC42H68O1317.73———
—P89Paniculatumoside A/paniculatumoside BC28H40O918.00———
—P90Glyasperin CC21H24O518.09———
—P91Ginsenoside F2C42H72O1318.57———
—P92Atractylenolide IIIC15H20O318.62———
—P93Sophoraisoflavone A/semilicoisoflavone BC20H16O619.91———
—P947-[4-(11-Hydroxy-undecyloxy)-phenyl]-7-pyridin-3-yl-hept-6-enoic acid ethyl esterC31H45NO420.82√—√
Total of prototypes 29 27 34
Metabolites Prototype Biotransformation Formula tR (min) Serum Urine Feces
M1P65Loss of C14H17NO3 + oxidationC10H11NO47.92—√—
M2P65Loss of C14H17NO3 + internal hydrolysisC10H13NO410.26—√—
M3P68DesaturationC10H16O415.84—√—
M4P68Loss of OC10H18O315.32—√—
M5P68Loss of O + hydrogenationC10H20O316.94√——
M6P65Loss of C14H18N2O3 + ketone formationC10H8O412.16—√—
M7P27Loss of O + glucuronidationC13H14O910.48—√—
P31Glucuronidation
M8P47Loss of C27H38O16 + ketone formationC13H16O711.22√√—
M9P47Loss of C27H38O16 and OC13H18O515.34—√—
M10P28Loss of O and C7H4O5 + hydrogenationC13H18O88.74—√—
P47Loss of C27H38O15 + oxidation
M11P25Loss of CH2C13H19NO46.13—√—
M12P25Loss of CH2 + sulfate conjugationC13H19NO7S9.85—√—
M13P63Loss of C26H28O13 + glutamine conjugationC14H16N2O413.77—√—
M14P63Loss of C21H20O9 and OC14H16O513.34—√—
M15P63Loss of C21H20O8C14H16O712.21—√—
P26Loss of O and O + hydrogenation
M16P63Loss of C21H20O8 + oxidationC14H16O88.21—√—
M17P63Loss of C21H20O8 + oxidationC14H16O89.28—√—
M18P65Loss of C10H9NO3 + demethylation to carboxylic acidC14H17NO58.16—√—
M19P28Loss of C7H4O4+methylationC14H18O104.85—√—
M20—P28Loss of C7H4O4 + methylationC14H18O105.12—√—
M21—P28Loss of O and C7H4O5 + methylationC14H18O87.3—√—
P63Loss of C21H20O8 + internal hydrolysis
M22P28Loss of O and C7H4O5 + methylationC14H18O811.29—√—
P63Loss of C21H20O8 + internal hydrolysis
M23P65Loss of C10H9NO3C14H19NO315.44—√—
M24P65Loss of C10H9NO3C14H19NO315.73—√—
M25P25DesaturationC14H19NO414.65—√—
M26P65Loss of C10H8O3C14H20N2O37.03—√—
M27P65Loss of C10H8O3 + phosphorylationC14H21N2O6P5.86—√—
M28P25OxidationC14H21NO52.32——√
M29P25Sulfate conjugationC14H21NO7S12.22—√—
M30P25PhosphorylationC14H22NO7P10.82—√—
M31P43Loss of C12H20O9C15H10O714.63—√√
M32P44Loss of C11H18O9C15H12O414.47—√√
P63Loss of C20H24O11
M33P63Loss of C20H24O11 + oxidationC15H12O515.52—√—
M34P63Loss of C20H24O12 + internal hydrolysisC15H14O416.12—√√
M35P25MethylationC15H23NO414.47—√—
M36P47Loss of C13H16O7 and C13H16O6 + methylationC15H26O913.24—√—
M37P43Loss of C12H20O10 and O + methylationC16H12O518.12—√—
M38P65Loss of C14H18N2O3 + glucose conjugationC16H20O813.53—√—
M39P63Loss of C15H10O4 + internal hydrolysisC20H28O128.03—√—
M40P43Loss of C6H10O4 + oxidationC21H20O139.06—√—
M41P28Loss of O and O + methylationC21H22O128.03—√—
M42P63Loss of C9H6O2 + glucuronidationC32H38O199.94—√—
M43P59Loss of OC42H72O1315.11√√√
M44P27Loss of O and OC7H6O212.52—√—
P31Loss of O
M45P23Loss of O and OC7H6O313.51√√—
P27Loss of O
P28Loss of O and C13H14O10
P47Loss of C27H38O15 and C6H10O6 + demethylation to carboxylic acid
M46P23Loss of OC7H6O49.12—√—
P28Loss of C13H14O10
P31Oxidation
M47P27OxidationC7H6O54.16—√√
P28Loss of C13H14O9
M48P27Loss of O + sulfate conjugationC7H6O6S6.83√√—
P31Sulfate conjugation
M49P23Loss of O + sulfate conjugationC7H6O7S6.76√√—
P36Loss of C20H16O14 + sulfate conjugation
P27Sulfate conjugation
M50P27Loss of O and O + hydrogenationC7H8O28.01—√√
P31Loss of O + hydrogenation
P47Loss of C27H38O15 and C6H10O5
M51P27Loss of O and O + methylationC8H8O211.44—√—
P31Loss of O + methylation
M52P36Loss of C20H16O14 and O + methylationC8H8O313.29—√—
P26Loss of C7H8O5 and O + methylation
P23Loss of O and O + methylation
P27Loss of O + methylation
P31Methylation
M53P23Loss of O + methylationC8H8O412.58√√—
P27Methylation
P36Loss of C20H16O14 + methylation
P28Loss of C13H14O10 + methylation
M54P23MethylationC8H8O58.53√√√
P36Loss of C20H16O13 + methylation
P26Loss of C7H8O4 + methylation
P28Loss of C13H14O9 + methylation
M55P63Loss of C26H28O13 + internal hydrolysisC9H10O39√√—
M56P63Loss of C26H28O12C9H8O311.56√√√
P65Loss of CH2 and C14H18N2O3
M57P63Loss of C26H28O12 + oxidationC9H8O414.04——√
M58P63Loss of C26H28O12 + oxidationC9H8O49.04—√—
M59P50GlucuronidationC34H40O2210.68—√—
M60P37Ketone formationC33H38O229.37—√—
P43Glucuronidation
M61P47Loss of C13H16O7 and O + phosphorylationC27H41O17P5.05—√—
M62P50Demethylation to carboxylic acidC28H30O1810.57—√—
M63P59Loss of C6H10O6C36H62O821.05——√
P58Loss of C12H20O10
M64P37Loss of O and C6H10O6 + hydrogenationC27H32O1413.05——√
P43Loss of O and O + hydrogenation
M65P50Loss of C6H10O5 + demethylation to carboxylic acidC22H20O1310.6—√—
M66P84Loss of C12H16O12 + oxidationC30H46O519.88——√
P71Loss of C12H16O12 and C6H10O5 + oxidation
M67P84Loss of C12H16O12 + oxidationC30H46O519.47√—√
P71Loss of C12H16O12 and C6H10O5 + oxidation
M68P50Loss of C6H10O4C22H22O1213.16√√—
P37Loss of C12H20O9 + methylation
P43Loss of C6H10O4 + methylation
M69P37Loss of C12H20O10 + demethylation to carboxylic acidC21H18O1313.13√√—
P43Loss of C6H10O5 + demethylation to carboxylic acid
M70P50Loss of C6H10O4 and O + ketone formationC22H20O1212.83—√—
M71P50Loss of C6H10O4 and O + ketone formationC22H20O1212.51—√—
M72P84Loss of C12H16O12C30H46O422.29√——
P71Loss of C12H16O12 and C6H10O5
M73P84Loss of C12H16O13 + ketone formationC30H44O419.47√√√
M74P50Loss of C6H10O5C22H22O1115.04—√—
P37Loss of C12H20O10 + methylation
P43Loss of C6H10O5 + methylation
M75P50Loss of C6H10O5 + demethylation and methylene to ketoneC21H18O1213.1—√—
P37Loss of C12H20O10 + ketone formation
P43Loss of C6H10O5 + ketone formation
M76P50Loss of C6H10O5 + demethylation and methylene to ketoneC21H18O1212.79—√—
P37Loss of C12H20O10 + ketone formation
P43Loss of C6H10O5 + ketone formation
M77P50Loss of C6H10O4 and CH2OC21H20O1113.08√√—
P37Loss of C12H20O10
P43Loss of C6H10O5
M78P50Loss of C6H10O4 and CH2OC21H20O1112.95√√—
P37Loss of C12H20O10
P43Loss of C6H10O5
M79P44Loss of C5H8O4 + oxidationC21H22O1013.79—√—
P37Loss of O and C12H20O10 + hydrogenation
P43Loss of C6H10O5 and O + hydrogenation
M80P44Loss of C5H8O5 + demethylation to carboxylic acidC21H20O1011.83√√—
P50Loss of C6H10O5 and CH2O
P37Loss of O and C12H20O10
P43Loss of C6H10O5 and O
M81P44Loss of C5H8O5+ketone formationC21H20O910.5—√—
M82P44Loss of C5H8O5+hydrogenationC21H24O813.18—√—
M83P6Loss of H–2O + methylationC13H26O103.74—√—
M84P47Loss of C13H16O7 and C13H16O6 + demethylation and methylene to ketoneC13H20O107.5—√—
M85P58Loss of C36H60O9 + methylationC13H24O96.42—√—
P50Loss of C16H10O7 + methylation
P37Loss of C21H18O12 + methylation
P7Loss of C6H10O6 and O + methylation
P43Loss of C15H8O7 + methylation
P6Loss of O and O + methylation
P8Loss of C6H10O6 and C6H10O6 + methylation
M86P47Loss of C27H38O15 + methylationC14H20O711.44—√—
P74Loss of C21H28O10 + methylation
M87P50Loss of C12H20O9 and CH2OC15H10O613.08—√—
P37Loss of C18H30O15
P43Loss of C12H20O10
M88P47Loss of C27H38O16C13H18O610.65—√√
P74Loss of C21H28O11
M89P47Loss of C27H38O16 + loss of hydroxymethyleneC12H16O511.17—√—
P74Loss of C21H28O11 + loss of hydroxymethylene
M90P44Loss of C11H18O10C15H12O310.5—√—
M91P12Loss of O + loss of hydroxymethyleneC9H11N5O21.38——√
M92P74Loss of C13H16O6 and C13H16O6C8H14O511.2—√—
M93P36Loss of C20H16O13 + decarboxylationC6H6O36—√—
M94P26Loss of C7H4O5C7H10O414.59—√—
M95P36Loss of C20H16O14 + taurine conjugationC9H11NO6S2.81√——
M96P36Loss of C14H6O10 + demethylation and methylene to ketoneC12H12O94.71—√—
M97P23Loss of O and O + glucose conjugationC13H16O84.11—√—
P36Loss of C14H6O10
P26Loss of C7H8O5 and O + glucose conjugation
P31Glucose conjugation
P47Loss of C27H38O16 + demethylation to carboxylic acid
M98P36Loss of C14H6O10 + hydrogenationC13H18O88.74—√—
M99P36Loss of C13H12O10C14H10O813.63—√—
M100P23Loss of O + glucose conjugationC13H16O96.81√√—
P27Glucose conjugation
P28Loss of C7H4O5
P36Loss of C14H6O9
P26Loss of C7H8O5 + glucose conjugation
M101P23Loss of O + glucose conjugationC13H16O96.57√√—
P27Glucose conjugation
P28Loss of C7H4O5
P36Loss of C14H6O9
P26Loss of C7H8O5 + glucose conjugation
M102P23Loss of O + glucuronidationC13H14O105.69—√—
P26Loss of C7H8O5 + glucuronidation
P27Glucuronidation
P28Loss of C7H4O5 + ketone formation
M103P36Loss of C14H6O9 + methylationC14H18O93.63—√—
M104P13Loss of H–2O + hydrogenationC14H16O107.96—√—
M105P36Loss of C13H12O8 + methylationC15H12O1011.93—√—
M106P36Loss of C13H12O8 + methylationC15H12O1010.7—√—
M107P36Loss of C13H12O9 + glutamine conjugationC19H18N2O1114.07——√
Total of metabolites 22 96 18
P2 arginine [54-56], P5 trigonelline [57], P59 ginsenoside Rg1 [58], P69 isoliquiritigenin [59], P82 ginsenoside Rd [60, 61], and P84 glycyrrhizic acid [62-64] would alleviate the symptom of UC based on anti-inflammation or antioxidant activities. Besides, P15 isoleucine [65], P17 uridine [12, 66], P21 guanosine [67], P23 gallic acid [68, 69], P43 rutin [70, 71], P51 vanillic acid [72], P70 quercetin [73, 74], P72 ginsenoside Rb1 [75], and P81 betulin [76]were confirmed to treat UC through NF-κB pathway. P8 stachyose increased beneficial microbiota and bacterial diversity to alleviate colitis mice [77]. P45 hyperoside ameliorates ulcer colitis mice through MKRN1-mediated regulation of PPARγ signaling and Th17/Treg balance [78]. The effect of those metabolisms on UC was worth to study for new drug development.
  78 in total

1.  New maltol glycosides from Flos Sophorae.

Authors:  Yi Zhang; Lu Qu; Lili Liu; Xiaoxia Li; Erwei Liu; Lifeng Han; Shiming Fang; Xiumei Gao; Tao Wang
Journal:  J Nat Med       Date:  2014-11-15       Impact factor: 2.343

2.  TCM: Made in China.

Authors:  Felix Cheung
Journal:  Nature       Date:  2011-12-21       Impact factor: 49.962

3.  Active constituents from Sophora japonica exhibiting cellular tyrosinase inhibition in human epidermal melanocytes.

Authors:  Yuan-Hsin Lo; Rong-Dih Lin; Yi-Pei Lin; Yan-Ling Liu; Mei-Hsien Lee
Journal:  J Ethnopharmacol       Date:  2009-05-05       Impact factor: 4.360

4.  Deciphering chemical interactions between Glycyrrhizae Radix and Coptidis Rhizoma by liquid chromatography with transformed multiple reaction monitoring mass spectrometry.

Authors:  Zhenhao Li; Ting Liu; Jie Liao; Ni Ai; Xiaohui Fan; Yiyu Cheng
Journal:  J Sep Sci       Date:  2017-02-27       Impact factor: 3.645

5.  Global identification of chemical constituents and rat metabolites of Si-Miao-Wan by liquid chromatography-electrospray ionization/quadrupole time-of-flight mass spectrometry.

Authors:  Jin-Jin Lu; Xue-Wen Hu; Ping Li; Jun Chen
Journal:  Chin J Nat Med       Date:  2017-07

6.  Saponins from European Licorice Roots ( Glycyrrhiza glabra).

Authors:  Christian Schmid; Corinna Dawid; Verena Peters; Thomas Hofmann
Journal:  J Nat Prod       Date:  2018-07-31       Impact factor: 4.050

7.  Streptococcus mutans sortase A inhibitory metabolites from the flowers of Sophora japonica.

Authors:  Woo-Young Yang; Tae Hyung Won; Chan-Hong Ahn; So-Hyoung Lee; Hyeong-Cheol Yang; Jongheon Shin; Ki-Bong Oh
Journal:  Bioorg Med Chem Lett       Date:  2015-02-27       Impact factor: 2.823

8.  Structural characterization and identification of flavonoid aglycones in three Glycyrrhiza species by liquid chromatography with photodiode array detection and quadrupole time-of-flight mass spectrometry.

Authors:  Shiqi Fang; Qiyang Qu; Yunfeng Zheng; Huanhuan Zhong; Chenxiao Shan; Fang Wang; Cunyu Li; Guoping Peng
Journal:  J Sep Sci       Date:  2016-05-13       Impact factor: 3.645

9.  Metabolite profiling of polyphenols in a Terminalia chebula Retzius ayurvedic decoction and evaluation of its chemopreventive activity.

Authors:  Federica Pellati; Renato Bruni; Davide Righi; Alessandro Grandini; Massimilano Tognolini; Francesco Pio Prencipe; Ferruccio Poli; Stefania Benvenuti; Daniele Del Rio; Damiano Rossi
Journal:  J Ethnopharmacol       Date:  2013-03-15       Impact factor: 4.360

10.  Rutin has intestinal antiinflammatory effects in the CD4+ CD62L+ T cell transfer model of colitis.

Authors:  Cristina Mascaraque; Carlos Aranda; Borja Ocón; María Jesús Monte; María Dolores Suárez; Antonio Zarzuelo; José Juan García Marín; Olga Martínez-Augustin; Fermín Sánchez de Medina
Journal:  Pharmacol Res       Date:  2014-10-02       Impact factor: 7.658
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